Dissection of transcriptional events in incompatible reactions of ‘Bearss’ lemon (Citrus limon) and ‘Valencia’ sweet orange (C. sinensis) on a novel citrandarin (C. reticulata × Poncirus trifoliata) rootstock. Dissection of transcriptional events in incompatible reactions of ‘Bearss’ lemon (Citrus limon) and ‘Valencia’ sweet orange (C. sinensis) on a novel citrandarin (C. reticulata × Poncirus trifoliata) rootstock

Citrus and most other fruit crops are commercially propagated via grafting, which ensures trees have consistent fruit traits combined with favorable traits from the rootstock such as soil adaptability, vigor, and resistance to soil pathogens. Breeding new rootstocks requires careful agronomic evaluations, and widespread use of new rootstocks and scions requires graft compatibility with commercially important scions and rootstocks. Graft incompatibility can occur when the scion and rootstock are not able to form a permanent, healthy union. Understanding and preventing graft incompatibility is therefore of paramount importance in the breeding of new fruit cultivars and in the choice of scion and rootstock by growers. The rootstock US-1283 is a citrandarin generated from a cross of ‘Ninkat’ mandarin (Citrus reticulata) and ‘Gotha Road’ #6 trifoliate orange (Poncirus trifoliata). It was released in 2014 after years of field evaluation because of its superior productivity and good fruit quality on ‘Hamlin’ sweet orange (C. sinensis) under Florida’s growing conditions. Subsequently, it was observed that trees of ‘Bearss’ lemon (C. limon) and ‘Valencia’ sweet orange (C. sinensis) grafted onto US-1283 exhibited apparent incompatible and unhealthy growth near the graft union. The incompatibility manifested as stem grooving and necrosis underneath the bark on the rootstock side of the graft. A genetically similar citrandarin rootstock, US-812 (C. reticulata ‘Sunki’ × P. trifoliata ‘Benecke’), is fully graft compatible with the same scions. Transcriptome analysis was performed on the vascular tissues above and below the graft union of compatible US-812 and incompatible US-1283 graft combinations with ‘Bearss’ and ‘Valencia’ to identify expression networks associated with incompatibility and help understand the processes and potential causes of incompatibility in citrus. Transcriptional reprogramming was stronger in the incompatible rootstock than in the grafted scions. Functional analysis of the transcriptional events below the graft unions of US-1283 incompatible combinations revealed differentially expression genes (DEGs) associated with oxidative stress and plant defense, among other pathways, similar to a pathogen-induced immune response localized to the rootstock, although no known pathogens were detected in the assayed plants. These changes were not observed above the graft unions.Differentially expressed genes (DEGs) in US-1283, but not the scions, were associated with oxidative stress and plant defense, among others, similar to a pathogen-induced immune response localized to the rootstock. No pathogen infection was detected. It is hypothesized this response could have been triggered by signaling miscommunications between rootstock and scion either through 1) unknown molecules from the scion that were perceived as danger signals by the rootstock, 2) missing signals from the scion or missing receptors in the rootstock necessary for the formation of a healthy graft union, 3) the overall perception of the scion by the rootstock as non-self, or 4) a combination of the above. Overall design: Our objective was to identify pathways, expression networks, and molecular mechanisms associated with incompatibility by analyzing transcriptome changes in rootstocks with similar genetic backgrounds but different responses to grafting. Given the increased numbers of novel scions and rootstocks developed by citrus breeders and the importance of grafting for propagation, there is an urgent need to develop effective and fast tools to understand and diagnose graft incompatibility and prevent consequent unexpected tree loss in the nursery and the field

柑橘及绝大多数其他果树均通过嫁接（grafting）进行商业化繁殖，该手段可确保植株兼具稳定的果实性状与砧木（rootstock）的优良特性，如土壤适应性、生长势以及对土壤病原菌的抗性。培育新型砧木需开展严谨的农艺学评价，而新型砧木与接穗（scion）的大规模推广应用，则要求其与商业化重要接穗及砧木具备嫁接亲和性。当接穗与砧木无法形成持久健康的接合部时，便会出现嫁接不亲和（graft incompatibility）。因此，解析并防控嫁接不亲和问题，对新型果树品种培育以及种植者选择接穗与砧木均至关重要。

砧木US-1283是由‘Ninkat’橘（Citrus reticulata）与‘Gotha Road’#6枳（Poncirus trifoliata）杂交得到的枳橙（citrandarin）。经多年田间评价后，该砧木于2014年获批推广，因其在佛罗里达州种植条件下，搭配‘Hamlin’甜橙（C. sinensis）时表现出优异的丰产性与良好的果实品质。后续研究发现，以‘Bearss’柠檬（C. limon）与‘Valencia’甜橙（C. sinensis）为接穗、嫁接至US-1283砧木的植株，在嫁接接合部附近表现出明显的不亲和与生长异常。该不亲和现象表现为嫁接接合部砧木侧的树皮下方出现茎沟纹与坏死。遗传背景与US-1283相近的枳橙砧木US-812（C. reticulata ‘Sunki’ × P. trifoliata ‘Benecke’），则与上述接穗完全亲和。

研究团队以‘Bearss’柠檬与‘Valencia’甜橙为接穗，分别嫁接至亲和性砧木US-812与不亲和性砧木US-1283，对二者嫁接接合部上下方的维管组织（vascular tissues）开展转录组（transcriptome）分析，以期鉴定与嫁接不亲和相关的表达网络，助力解析嫁接不亲和的发生过程与潜在诱因。研究发现，不亲和砧木中的转录重编程（transcriptional reprogramming）程度强于接穗。对US-1283不亲和组合中嫁接接合部下方的转录事件进行功能分析后发现，差异表达基因（differentially expressed genes, DEGs）除参与其他通路外，还与氧化应激（oxidative stress）及植物防御（plant defense）相关，其特征类似于局限于砧木的病原菌诱导免疫应答（immune response），尽管受试植株未检测到已知病原菌。上述变化仅出现在嫁接接合部下方，接穗侧未观察到类似改变。

US-1283砧木中检测到的差异表达基因（而非接穗），确实与氧化应激、植物防御等通路相关，其特征类似于局限于砧木的病原菌诱导免疫应答，且未检测到病原菌感染。研究人员据此提出假说：该应答可能由接穗与砧木间的信号交流紊乱触发，具体可能包括以下4种情形：1）接穗产生的未知分子被砧木识别为危险信号；2）接穗缺失必要信号，或砧木缺少形成健康嫁接接合部所需的受体；3）砧木将接穗识别为异源物质；4）上述多种情形共同作用。

本研究的整体实验设计目标为：通过分析遗传背景相近但嫁接响应不同的砧木的转录组变化，鉴定与嫁接不亲和相关的通路、表达网络及分子机制。鉴于柑橘育种者培育的新型接穗与砧木数量日益增多，且嫁接繁殖手段的重要性愈发凸显，当前亟需开发高效便捷的工具，以解析、诊断嫁接不亲和问题，并规避苗圃与大田生产中由此引发的非预期植株损失。